The renin-angiotensin system (RAS) plays a major role in the regulation of blood pressure and electrolyte balance in humans and other mammals. Although the RAS has been intensively examined at the physiological level the molecular mechanisms regulating the expression of genes in the system are only beginning to be uncovered. With genetic studies suggesting a link between the RAS genes and hypertension in animals and in humans, and transgenic studies demonstrating the potential for abnormalities in the RAS to cause hypertension, gaining a clear understanding of these important mechanisms becomes essential. Our strategy to investigate the molecular mechanisms regulating HuRen gene expression is two pronged. First, Calu-6 and As4.1 cells will be used for transfection analysis and as a source of transcription factors for examining DNA-protein interactions. Second, transgenic mice will be used as a model to investigate tissue-specific and cell-specific expression, and physiologic and pharmacologic regulation of the gene. We hypothesize that basal transcriptional regulation of the HuRen gene is conferred by a group of closely clustered weak regulatory elements within the 5' flanking region and a silencer present within the first intron. High level expression of the gene is controlled by a gene-specific enhancer. Inducibility by increased intracellular cAMP in Calu-6 cells occurs largely by a post- transcriptional mechanism involving a decrease in HuRen mRNA turnover leading to enhanced message stability. Tissue and cell-specificity of the gene in transgenic mice is conferred by regions located in the proximal promoter, the enhancer, and within the body of the gene itself. In order to further test these hypotheses we propose to: 1) identify the specific DNA sequences and transcription factor contact sites regulating basal transcriptional activity of the HuRen gene, 2) characterize the HuRen enhancer sequence, 3) identify sequences regulating appropriate tissue- and cell-specificity of the HuRen gene in transgenic mice, and 4) investigate the mechanisms causing the cAMP-mediated increase in endogenous HuRen mRNA in Calu-6 cells. The use of bonafide renin expressing cell lines and transgenic animals will provide us with unparalleled tools that will result in meaningful and physiologically relevant data.
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